A. Field Of The Invention
The present invention relates to recombinant DNA technology. It is especially useful for allowing the production of Epstein-Barr virus vectors capable of carrying foreign genes into B-lymphocytes.
B. Description Of The Art
Although some progress has been made in using recombinant-DNA techniques to carry foreign genes into certain eukaryotic hosts (e.g. using retrovirus vectors), it is of particular interest to develop vector systems that are specially suited for use with B-lymphocytes. Apart from pure research applications, this would enhance the ability to produce foreign proteinaceous materials in B-lymphocytes (e.g. where lymphocytes are a host of choice), and/or permit the development of drugs that interact with B-lymphocytes.
EBV is a human herpes virus of the gamma herpesviridae subfamily that infects and immortalizes B-lymphocytes in vitro. It is one of the lymphotrophic herpes viruses. Its viral DNA is usually maintained as complete multiple copies of plasmids in these immortalized cells. An origin of plasmid replication, oriP, is the only element required in cis for EBV plasmid replication. This was the subject of U.S. Pat. No. 4,686,186 (the disclosure of this patent and of all articles referred to herein are incorporated by reference as if fully set forth). These immortalized cells are said to be "latently" infected by EBV because only a few viral genes are usually expressed in them, and virion structural (packaging) genes are not among these normally expressed genes.
Under unusual circumstances (e.g. induction) viral gene expression can change dramatically to yield the "lytic" phase of the EBV life cycle. Most or all of the 90-100 viral genes are expressed during the lytic phase, and the viral DNA is amplified by a replication mechanism distinct from that used to maintain the viral DNA during its latent phase of infection.
Because of these factors, EBV is of interest as a potential vector system. However, there exists in nature no identified host cell which normally supports a lytic infection by this natural herpes virus. Moreover, the viral DNA (which is 172 kbp in length) is too large to permit engineering in vitro with the goal of introducing the mutant genomes into recipient cells. Also, primary human B-lymphocytes which are the natural host for infection with EBV are both intractable in culture and recalcitrant to DNA transfection. Thus, it can be seen that a need has existed for developing means of using EBV recombinants to carrying foreign genes into eukaryotic cells (such as B-lymphocytes).